1. Field of the Invention
The present invention relates to methods for improving a genetic stability of a foreign insert nucleotide sequence in a recombinant RNA virus and recombinant RNA viruses comprising a foreign insert nucleotide sequence with improved genetic stability. More particularly, the present invention relates to (a) methods for improving a genetic stability of a foreign insert nucleotide sequence in a recombinant RNA virus, (b) a recombinant single-stranded RNA virus comprising a foreign insert nucleotide sequence with improved genetic stability, (c) a recombinant poliovirus comprising a foreign insert nucleotide sequence with improved genetic stability, (d) methods for construction of sequence-adjusted exogeneous nucleotide or artificially synthesized foreign inserts nucleotide by template/ligation-free PCR, and (e) a vaccine composition comprising a recombinant single-stranded RNA virus.
2. Description of the Related Art
Live attenuated viral vaccines have been reported to have several advantages over other types of vaccines: low cost for production, higher immunogenicity, and easy for administration. However, the greatest advantage has been offered by the well-characterized molecular structures of target viruses which enable investigators to manipulate the viral cDNA genome with a recombinant DNA technique even with RNA viruses, as to produce recombinant progeny viruses (Rolph, M. S. and I. A. Ramshaw., Curr. Opion. In Immunology 9:517-524(1997)). The principal idea is to insert the exogenous insert nucleotide sequence encoding the desired foreign antigen into the attenuated viral genome without altering the viability of the virus. Theoretically, recombinant viruses can be used as an efficient recombinant vaccine, since the inserted genes can be replicated, expressed and packaged along with the viral genome, subsequently leading to induce immune responses not only to the parental viruses but also to the introduced foreign antigens.
The utility of this vaccine approach, however, has been largely constrained by several factors such as a limitation of an insert size, far reduced replication capacity, genetic instability, or a recurrence of the pathogenicity of the parental or recombinant viruses.
Many attempts have been made to manipulate the poliovirus (PV) as a favorable vaccine vector because of its attractive characteristics of safe usage, low cost, convenient administration, and long-lasting protective immunity in both mucosal and systemic immune responses, which have been established for decades. However, One of the most serious obstacles for a wide application of recombination poliovirus as an effective live viral vaccine vector has been the genetic instability of the recombinant virus.
Poliovirus, as a member of Picornaviridae, is a nonenveloped, positive-sense single-stranded RNA virus containing 7.44 kb of RNA genome. The genome contains an internal ribosomal entry site (IRES) followed by a single open reading frame (ORF) encoding a long polyprotein. The IRES element controls the expression of the polyprotein that is subsequently cleaved into several structural and nonstructural proteins by three kinds of virus-encoded proteases (2Apro, 3Cpro, and 3CDPro). A major viral protease, 3Cpro, and its precursor, 3CDpro, cleave the polyprotein at a specific site (AXXQ/G) within the expressed polyprotein, while a minor protease, 2Apro, cleaves the polyprotein at the junction between the P1 and P2 regions. Exactly 60 copies of each of four different capsid proteins (VP1, VP2, VP3, and VP4) are assembled into a rigid icosahedral viral capsid that concomitantly encapsidates the viral genome.
The polyprotein fusion strategy, one of the strategies for poliovirus-based vaccine developments, was directed at fusing the foreign insert to either at N-terminus or at the junction between the capsid proteins and nonstructural proteins (P1/P2) in the long polyprotein with an artificial cleavage site for poliovirus-specific proteases (Andino, R., D. et al., Science 265:1448-1451(1994) and U.S. Pat. No. 5,965,124). Accordingly, the foreign insert is cleaved-off by one of proteases and remains as a free form in the cytoplasm after being translated together with the viral proteins. A number of Mahoney-vector-based recombinant polioviruses were constructed by this strategy, and were demonstrated for their humoral, cellular, or mucosal immunogenicity against introduced exogenous antigens (Crotty, S., et al, J. Virol. 73:9485-9495(1999); and Mandl, S. et al., Proc. Natl. Acad. Sci, USA 95:8216-8221(1998)).
However, the plausibility of this strategy was challenged by the genetic instability of the recombinant viruses (Tang, S., et al, J. Virol. 71:7841-7850(1997); Mueller, S., and E. Wimmer., J. Virol. 72:20-31(1998)). Previous reports have suggested that the genetic instability of the rec-PV would be associated with the insert size limitation and/or genetic recombination within intra- (Tang, S., et al, J. Virol. 71:7841-7850(1997)) or between inter-sequences during minus-strand synthesis (Mueller, S., and E. Wimmer., J. Virol. 72:20-31(1998); Wimmer, E., et al, Annu. Rev. Genet. 27:353-436(1993)). Nevertheless, a clear molecular mechanism controlling insert stability has not been well established.
Consequently, there is a need of a novel strategy to overcome the shortcomings of the poliovirus vector systems aforementioned.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
To be free from shortcomings of the poliovirus vector systems, in particular, genetic instability of the foreign insert integrated into the recombinant polioviruses, the present inventors have examined potential factors governing genetic stability of foreign insert by constructing and exploiting many different recombinant polioviruses, which contain a series of different original or sequence-adjusted foreign inserts. From these experiments, we have accomplished present invention that i) the insert genetic stability is strongly associated with the G/C contents and its distribution patterns within the size limitation, and ii) the insert genetic stability can be markedly enhanced by increasing the G/C contents of the foreign insert.
Accordingly, it is an object of this invention to provide a method for improving a genetic stability of a foreign insert nucleotide sequence in a recombinant single-stranded RNA virus vector.
It is another object of this invention to provide a method for constructing a recombinant single-stranded RNA virus containing a foreign insert nucleotide sequence with improved genetic stability.
It is still another object of this invention to provide a recombinant single-stranded RNA virus comprising a foreign insert nucleotide sequence with improved genetic stability.
It is further object of this invention to provide a recombinant poliovirus comprising a foreign insert nucleotide sequence with improved genetic stability.
It is still further object of this invention to provide a method for construction of sequence-adjusted or artificially synthesized foreign inserts using template/ligation-free PCR method.
It is another object of this invention to provide a vaccine composition comprising a recombinant single-stranded RNA virus.
Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.